Mentoring Minority Cancer Researchers of Tomorrow: Comparison of the Face-to-Face, Virtual, and Hybrid Training Methods of the CaRE2 Summer Cancer Research Education and Training Program.

In our previous publication, we reported a framework to develop an undergraduate cancer research training program at Florida A&M University (FAMU) under the umbrella of the Florida-California Cancer Research, Education, and Engagement (CaRE2) Health Equity Center activity by harnessing the resources available at FAMU, the University of Florida (UF), and the University of Southern California (USC) Cancer Centers. The implementation of the CaRE2 face-to-face training platform was dramatically affected by the COVID-19 pandemic during the summer of 2020 and 2021 training periods. However, a concerted effort was made to restructure the face-to-face training model into virtual and hybrid training methods to maintain the continuity of the program during the pandemic. This article compared the three methods to identify the best platform for training URM students in cancer disparity research. The program's effectiveness was measured through motivation, experiences, and knowledge gained by trainees during and one year after the completion of the program. The results showed that the participants were highly positive in their feedback about the professional and academic values of the program. Although the virtual and hybrid methods experienced significant challenges during the pandemic, the hybrid training module offered an "above average" effectiveness in performance, like the face-to-face mentoring platform in mentoring URM students in cancer disparity research.

Lack of racial and ethnic diversity in lung cancer cell lines contributes to lung cancer health disparities.

Lung cancer is the leading cause of cancer death in the United States and worldwide, and a major source of cancer health disparities. Lung cancer cell lines provide key in vitro models for molecular studies of lung cancer development and progression, and for pre-clinical drug testing. To ensure health equity, it is imperative that cell lines representing different lung cancer histological types, carrying different cancer driver genes, and representing different genders, races, and ethnicities should be available. This is particularly relevant for cell lines from Black men, who experience the highest lung cancer mortality in the United States. Here, we undertook a review of the available lung cancer cell lines and their racial and ethnic origin. We noted a marked imbalance in the availability of cell lines from different races and ethnicities. Cell lines from Black patients were strongly underrepresented, and we identified no cell lines from Hispanic/Latin(x) (H/L), American Indian/American Native (AI/AN), or Native Hawaiian or other Pacific Islander (NHOPI) patients. The majority of cell lines were derived from White and Asian patients. Also missing are cell lines representing the cells-of-origin of the major lung cancer histological types, which can be used to model lung cancer development and to study the effects of environmental exposures on lung tissues. To our knowledge, the few available immortalized alveolar epithelial cell lines are all derived from White subjects, and the race and ethnicity of a handful of cell lines derived from bronchial epithelial cells are unknown. The lack of an appropriately diverse collection of lung cancer cell lines and lung cancer cell-of-origin lines severely limits racially and ethnically inclusive lung cancer research. It impedes the ability to develop inclusive models, screen comprehensively for effective compounds, pre-clinically test new drugs, and optimize precision medicine. It thereby hinders the development of therapies that can increase the survival of minority and underserved patients. The noted lack of cell lines from underrepresented groups should constitute a call to action to establish additional cell lines and ensure adequate representation of all population groups in this critical pre-clinical research resource.

Florida-California Cancer Research, Education and Engagement (CaRE2) Health Equity Center: Structure, Innovations, and Initial Outcomes.

INTRODUCTION: The Florida-California Cancer Research, Education, and Engagement (CaRE2) Health Equity Center is a triad partnership committed to increasing institutional capacity for cancer disparity research, the diversity of the cancer workforce, and community empowerment. This article provides an overview of the structure, process innovations, and initial outcomes from the first 4 years of the CaRE2 triad partnership. METHODS: CaRE2 serves diverse populations in Florida and California using a "molecule to the community and back" model. We prioritize research on the complex intersection of biological, environmental, and social determinants health, working together with scientific and health disparities communities, sharing expertise across institutions, bidirectional training, and community outreach. Partnership progress and outcomes were assessed using mixed methods and four Program Steering Committee meetings. RESULTS: Research capacity was increased through development of a Living Repository of 81 cancer model systems from minority patients for novel cancer drug development. CaRE2 funded 15 scientific projects resulting in 38 publications. Workforce diversity entailed supporting 94 cancer trainees (92 URM) and 34 ESIs (32 URM) who coauthored 313 CaRE2-related publications and received 48 grants. Community empowerment was promoted via outreaching to more than 3000 individuals, training 145 community cancer advocates (including 28 Community Scientist Advocates), and publishing 10 community reports. CaRE2 members and trainees together have published 639 articles, received 61 grants, and 57 awards. CONCLUSION: The CaRE2 partnership has achieved its initial aims. Infrastructure for translational cancer research was expanded at one partner institution, and cancer disparities research was expanded at the two cancer centers.

Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis.

Lung cancer is the leading cause of cancer mortality worldwide and KRAS is the most commonly mutated gene in lung adenocarcinoma (LUAD). The 78-kDa glucose-regulated protein GRP78/BiP is a key endoplasmic reticulum chaperone protein and a major pro-survival effector of the unfolded protein response (UPR). Analysis of the Cancer Genome Atlas database and immunostain of patient tissues revealed that compared to normal lung, GRP78 expression is generally elevated in human lung cancers, including tumors bearing the KRASG12D mutation. To test the requirement of GRP78 in human lung oncogenesis, we generated mouse models containing floxed Grp78 and Kras Lox-Stop-Lox G12D (KrasLSL-G12D) alleles. Simultaneous activation of the KrasG12D allele and knockout of the Grp78 alleles were achieved in the whole lung or selectively in lung alveolar epithelial type 2 cells known to be precursors for adenomas that progress to LUAD. Here we report that GRP78 haploinsufficiency is sufficient to suppress KrasG12D-mediated lung tumor progression and prolong survival. Furthermore, GRP78 knockdown in human lung cancer cell line A427 (KrasG12D/+) leads to activation of UPR and apoptotic markers and loss of cell viability. Our studies provide evidence that targeting GRP78 represents a novel therapeutic approach to suppress mutant KRAS-mediated lung tumorigenesis.

The Sulfotransferase SULT1C2 Is Epigenetically Activated and Transcriptionally Induced by Tobacco Exposure and Is Associated with Patient Outcome in Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer-related death. Tobacco exposure is associated with 80-90% of lung cancer cases. The SULT1C2 sulfotransferase modifies xenobiotic compounds to enhance secretion but can also render these compounds carcinogenic. To determine if SULT1C2 contributes to tobacco-related carcinogenesis in the lung, we analyzed the expression and epigenetic state of SULT1C2 in human lung adenocarcinoma (LUAD) samples and in LUAD cell lines exposed to cigarette smoke condensate (CSC). SULT1C2 expression was significantly positively correlated to overall LUAD patient survival in smokers, was elevated in LUAD tumors compared to adjacent non-tumor lung, and was significantly correlated with levels of patient exposure to tobacco smoke. SULT1C2 promoter DNA methylation was inversely correlated with expression in LUAD, and hypomethylation of the SULT1C2 promoter was observed in Asian patients, as compared to Caucasians. In vitro analysis of LUAD cell lines indicates that CSC stimulates expression of SULT1C2 in a dose-dependent and cell-line-specific manner. In vitro methylation of the SULT1C2 promoter significantly decreased transcriptional activity of a reporter plasmid, and SULT1C2 expression was activated by the DNA demethylating agent 5-Aza-2'-deoxycytidine in a cell line in which the SULT1C2 promoter was hypermethylated. An aryl hydrocarbon receptor (AHR) binding site was detected spanning critical methylation sites upstream of SULT1C2. CSC exposure significantly increased AHR binding to this predicted binding site in the SULT1C2 promoter in multiple lung cell lines. Our data suggest that CSC exposure leads to activation of the AHR transcription factor, increased binding to the SULT1C2 promoter, and upregulation of SULT1C2 expression and that this process is inhibited by DNA methylation at the SULT1C2 locus. Additionally, our results suggest that the level of SULT1C2 promoter methylation and gene expression in normal lung varies depending on the race of the patient, which could in part reflect the molecular mechanisms of racial disparities seen in lung cellular responses to cigarette smoke exposure.

Developing a Novel Framework for an Undergraduate Cancer Research Education and Engagement Program for Underrepresented Minority Students: the Florida-California CaRE2 Research Education Core (REC) Training Program.

Lack of substantive research experiences and technical skills mentoring during undergraduate studies leaves many underrepresented minority (URM) students unprepared to apply to competitive graduate programs. As a part of our ongoing effort to increase the pipeline for the development and training of successful URM scientists in biomedical sciences with focus on reducing cancer health disparities, the Florida-California Cancer Research Education and Engagement (CaRE2) Health Equity Center was launched in 2018. Funded through an NIH/NCI U54 grant mechanism, the CaRE2 Center is a triad partnership among Florida Agricultural and Mechanical University (FAMU), a minority-serving institution, University of Florida (UF), and University of Southern California (USC) Cancer Center. One of the objectives of the triad partnership is to promote the coordination and implementation of the training of the next generation of Black and Latinx biomedical scientists in Florida and California. An important component of the CaRE2 program is the Research and Education Core (REC) designed to coordinate the training of URM students and researchers at different levels in their academic and professional developments. The undergraduate cancer research training program under FAMU-CaRE2 Center is a 3-year (2018-2021) project to identify, train, mentor, and provide the URM undergraduate students with the support network they need to flourish in the program and beyond. In its year-1 funding cycle, the program has made significant progress in developing a novel framework for an undergraduate cancer research education and engagement program at FAMU, one of the forefront minority institutions in the nation. The mentored research program is complemented with professional development and engagement activities, including cancer research seminars, workshops, and community outreach activities. The purpose of this paper is to discuss the strategies implemented for an effective partnership, the leadership and mentoring skills, and outcomes from the year-1 experiences. In addition, we present the progress made in advancing the pool of underrepresented minority students with scientific and academic career progression paths focused on cancer health disparities.

TENET 2.0: Identification of key transcriptional regulators and enhancers in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related death and lung adenocarcinoma is its most common subtype. Although genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations driving lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We found over 32,000 enhancers that appear differentially activated between normal lung and lung adenocarcinoma. Among the identified transcriptional regulators inactivated in lung adenocarcinoma vs. normal lung, NKX2-1 was linked to a large number of silenced enhancers. Among the activated transcriptional regulators identified, CENPA, FOXM1, and MYBL2 were linked to numerous cancer-specific enhancers. High expression of CENPA, FOXM1, and MYBL2 is particularly observed in a subgroup of lung adenocarcinomas and is associated with poor patient survival. Notably, CENPA, FOXM1, and MYBL2 are also key regulators of cancer-specific enhancers in breast adenocarcinoma of the basal subtype, but they are associated with distinct sets of activated enhancers. We identified individual lung adenocarcinoma enhancers linked to CENPA, FOXM1, or MYBL2 that were associated with poor patient survival. Knockdown experiments of FOXM1 and MYBL2 suggest that these factors regulate genes involved in controlling cell cycle progression and cell division. For example, we found that expression of TK1, a potential target gene of a MYBL2-linked enhancer, is associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma epigenomes, highlighting novel potential targets for clinical intervention.

Genome-wide integration of microRNA and transcriptomic profiles of differentiating human alveolar epithelial cells.

The alveolar epithelium is comprised of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells, the latter being capable of self-renewal and transdifferentiation into AT1 cells for normal maintenance and restoration of epithelial integrity following injury. MicroRNAs (miRNAs) are critical regulators of several biological processes, including cell differentiation; however, their role in establishment/maintenance of cellular identity in adult alveolar epithelium is not well understood. To investigate this question, we performed genome-wide analysis of sequential changes in miRNA and gene expression profiles using a well-established model in which human AT2 (hAT2) cells transdifferentiate into AT1-like cells over time in culture that recapitulates many aspects of transdifferentiation in vivo. We defined three phases of miRNA expression during the transdifferentiation process as "early," "late," and "consistently" changed, which were further subclassified as up- or downregulated. miRNAs with altered expression at all time points during transdifferentiation were the largest subgroup, suggesting the need for consistent regulation of signaling pathways to mediate this process. Target prediction analysis and integration with previously published gene expression data identified glucocorticoid signaling as the top pathway regulated by miRNAs. Serum/glucocorticoid-regulated kinase 1 (SGK1) emerged as a central regulatory factor, whose downregulation correlated temporally with gain of hsa-miR-424 and hsa-miR-503 expression. Functional validation demonstrated specific targeting of these miRNAs to the 3'-untranslated region of SGK1. These data demonstrate the time-related contribution of miRNAs to the alveolar transdifferentiation process and suggest that inhibition of glucocorticoid signaling is necessary to achieve the AT1-like cell phenotype.

CLDN18.1 attenuates malignancy and related signaling pathways of lung adenocarcinoma in vivo and in vitro.

Claudins are a family of transmembrane proteins integral to the structure and function of tight junctions (TJ). Disruption of TJ and alterations in claudin expression are important features of invasive and metastatic cancer cells. Expression of CLDN18.1, the lung-specific isoform of CLDN18, is markedly decreased in lung adenocarcinoma (LuAd). Furthermore, we recently observed that aged Cldn18 -/- mice have increased propensity to develop LuAd. We now demonstrate that CLDN18.1 expression correlates inversely with promoter methylation and with LuAd patient mortality. In addition, when restored in LuAd cells that have lost expression, CLDN18.1 markedly attenuates malignant properties including xenograft tumor growth in vivo as well as cell proliferation, migration, invasion and anchorage-independent colony formation in vitro. Based on high throughput analyses of Cldn18 -/- murine lung alveolar epithelial type II cells, as well as CLDN18.1-repleted human LuAd cells, we hypothesized and subsequently confirmed by Western analysis that CLDN18.1 inhibits insulin-like growth factor-1 receptor (IGF-1R) and AKT phosphorylation. Consistent with recent data in Cldn18 -/- knockout mice, expression of CLDN18.1 in human LuAd cells also decreased expression of transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) and their target genes, contributing to its tumor suppressor activity. Moreover, analysis of LuAd cells in which YAP and/or TAZ are silenced with siRNA suggests that inhibition of TAZ, and possibly YAP, is also involved in CLDN18.1-mediated AKT inactivation. Taken together, these data indicate a tumor suppressor role for CLDN18.1 in LuAd mediated by a regulatory network that encompasses YAP/TAZ, IGF-1R and AKT signaling.

Positional integration of lung adenocarcinoma susceptibility loci with primary human alveolar epithelial cell epigenomes.

AIM: To identify functional lung adenocarcinoma (LUAD) risk SNPs. MATERIALS & METHODS: Eighteen validated LUAD risk SNPs (p ≤ 5 × 10-8) and 930 SNPs in high linkage disequilibrium (r2 > 0.5) were integrated with epigenomic information from primary human alveolar epithelial cells. Enhancer-associated SNPs likely affecting transcription factor-binding sites were predicted. Three SNPs were functionally investigated using luciferase assays, expression quantitative trait loci and cancer-specific expression. RESULTS: Forty-seven SNPs mapped to putative enhancers; 11 located to open chromatin. Of these, seven altered predicted transcription factor-binding motifs. Rs6942067 showed allele-specific luciferase expression and expression quantitative trait loci analysis indicates that it influences expression of DCBLD1, a gene that encodes an unknown membrane protein and is overexpressed in LUAD. CONCLUSION: Integration of candidate LUAD risk SNPS with epigenomic marks from normal alveolar epithelium identified numerous candidate functional LUAD risk SNPs including rs6942067, which appears to affect DCBLD1 expression. Data deposition: Data are provided in GEO record GSE84273.

Association of internal smoking dose with blood DNA methylation in three racial/ethnic populations.

BACKGROUND: Lung cancer is the leading cause of cancer-related death. While cigarette smoking is the primary cause of this malignancy, risk differs across racial/ethnic groups. For the same number of cigarettes smoked, Native Hawaiians compared to whites are at greater risk and Japanese Americans are at lower risk of developing lung cancer. DNA methylation of specific CpG sites (e.g., in AHRR and F2RL3) is the most common blood epigenetic modification associated with smoking status. However, the influence of internal smoking dose, measured by urinary nicotine equivalents (NE), on DNA methylation in current smokers has not been investigated, nor has a study evaluated whether for the same smoking dose, circulating leukocyte DNA methylation patterns differ by race. METHODS: We conducted an epigenome-wide association study (EWAS) of NE in 612 smokers from three racial/ethnic groups: whites (n = 204), Native Hawaiians (n = 205), and Japanese Americans (n = 203). Genome-wide DNA methylation profiling of blood leukocyte DNA was measured using the Illumina 450K BeadChip array. Average ß value, the ratio of signal from a methylated probe relative to the sum of the methylated and unmethylated probes at that CpG, was the dependent variables in linear regression models adjusting for age, sex, race (for pan-ethnic analysis), and estimated cell-type distribution. RESULTS: We found that NE was significantly associated with six differentially methylated CpG sites (Bonferroni corrected p < 1.48 × 10-7): four in or near the FOXK2, PBX1, FNDC7, and FUBP3 genes and two in non-annotated genetic regions. Higher levels of NE were associated with increasing methylation beta-valuesin all six sites. For all six CpG sites, the association was only observed in Native Hawaiians, suggesting that the influence of smoking dose on DNA methylation patterns is heterogeneous across race/ethnicity (p interactions < 8.8 × 10-8). We found two additional CpG sites associated with NE in only Native Hawaiians. CONCLUSIONS: In conclusion, internal smoking dose was associated with increased DNA methylation in circulating leukocytes at specific sites in Native Hawaiian smokers but not in white or Japanese American smokers.

Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Publisher Correction: Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

In the originally published version of this Article, the positions of the final two authors in the author list were inadvertently inverted during the production process. This error has now been corrected in both the PDF and HTML versions of the Article.

Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs.

The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call "entrapping". Our results support a key role for uc.339 in lung cancer.

Epigenome-wide analysis of DNA methylation in lung tissue shows concordance with blood studies and identifies tobacco smoke-inducible enhancers.

Smoking-associated DNA hypomethylation has been observed in blood cells and linked to lung cancer risk. However, its cause and mechanistic relationship to lung cancer remain unclear. We studied the association between tobacco smoking and epigenome-wide methylation in non-tumor lung (NTL) tissue from 237 lung cancer cases in the Environment And Genetics in Lung cancer Etiology study, using the Infinium HumanMethylation450 BeadChip. We identified seven smoking-associated hypomethylated CpGs (P < 1.0 × 10-7), which were replicated in NTL data from The Cancer Genome Atlas. Five of these loci were previously reported as hypomethylated in smokers' blood, suggesting that blood-based biomarkers can reflect changes in the target tissue for these loci. Four CpGs border sequences carrying aryl hydrocarbon receptor binding sites and enhancer-specific histone modifications in primary alveolar epithelium and A549 lung adenocarcinoma cells. A549 cell exposure to cigarette smoke condensate increased these enhancer marks significantly and stimulated expression of predicted target xenobiotic response-related genes AHRR (P = 1.13 × 10-62) and CYP1B1 (P < 2.49 × 10-61). Expression of both genes was linked to smoking-related transversion mutations in lung tumors. Thus, smoking-associated hypomethylation may be a consequence of enhancer activation, revealing environmentally-induced regulatory elements implicated in lung carcinogenesis.

Cross-Species Transcriptome Profiling Identifies New Alveolar Epithelial Type I Cell-Specific Genes.

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.

Data on isoaspartylation of neuronal ELAVL proteins.

This article contains experimental data examining the propensity of neuronal ELAVL proteins to become isoaspartylated. The data are related to the article "Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4" (M.A. Pulido, M.K. DerHartunian, Z. Qin, E.M. Chung, D.S. Kang, A.W. Woodham, J.A. Tsou, R. Klooster, O. Akbari, L. Wang, W.M. Kast, S.V. Liu, J.J.G.M. Verschuuren, D.W. Aswad, I.A. Laird-Offringa, 2016) [1], in which it was reported that the N-terminal region of recombinant human ELAVL4 protein, incubated under physiological conditions, acquires a type of highly immunogenic protein damage. Here, we present Western blot analysis data generated by using an affinity-purified polyclonal rabbit antibody (raised against an N-terminal ELAVL4 isoaspartyl-converted peptide) to probe recombinant protein fragments of the other three members of the ELAVL family: the highly homologous neuronal ELAVL2 (HuB) and ELAVL3 (HuC), and the much less homologous ubiquitously expressed ELAVL1 (HuR).

Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4.

Autoantibodies against SCLC-associated neuronal antigen ELAVL4 (HuD) have been linked to smaller tumors and improved survival, but the antigenic epitope and mechanism of autoimmunity have never been solved. We report that recombinant human ELAVL4 protein incubated under physiological conditions acquires isoaspartylation, a type of immunogenic protein damage. Specifically, the N-terminal region of ELAVL4, previously implicated in SCLC-associated autoimmunity, undergoes isoaspartylation in vitro, is recognized by sera from anti-ELAVL4 positive SCLC patients and is highly immunogenic in subcutaneously injected mice and in vitro stimulated human lymphocytes. Our data suggest that isoaspartylated ELAVL4 is the trigger for the SCLC-associated anti-ELAVL4 autoimmune response.

Pleiotropic Analysis of Lung Cancer and Blood Triglycerides.

Epidemiologically related traits may share genetic risk factors, and pleiotropic analysis could identify individual loci associated with these traits. Because of their shared epidemiological associations, we conducted pleiotropic analysis of genome-wide association studies of lung cancer (12 160 lung cancer case patients and 16 838 control subjects) and cardiovascular disease risk factors (blood lipids from 188 577 subjects, type 2 diabetes from 148 821 subjects, body mass index from 123 865 subjects, and smoking phenotypes from 74 053 subjects). We found that 6p22.1 (rs6904596, ZNF184) was associated with both lung cancer (P = 5.50x10(-6)) and blood triglycerides (P = 1.39x10(-5)). We replicated the association in 6097 lung cancer case patients and 204 657 control subjects (P = 2.40 × 10(-4)) and in 71 113 subjects with triglycerides data (P = .01). rs6904596 reached genome-wide significance in lung cancer meta-analysis (odds ratio = 1.15, 95% confidence interval = 1.10 to 1.21 ,: Pcombined = 5.20x10(-9)). The large sample size provided by the lipid GWAS data and the shared genetic risk factors between the two traits contributed to the uncovering of a hitherto unidentified genetic locus for lung cancer.

The 78-kD Glucose-Regulated Protein Regulates Endoplasmic Reticulum Homeostasis and Distal Epithelial Cell Survival during Lung Development.

Bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurity, has been linked to endoplasmic reticulum (ER) stress. To investigate a causal role for ER stress in BPD pathogenesis, we generated conditional knockout (KO) mice (cGrp78(f/f)) with lung epithelial cell-specific KO of Grp78, a gene encoding the ER chaperone 78-kD glucose-regulated protein (GRP78), a master regulator of ER homeostasis and the unfolded protein response (UPR). Lung epithelial-specific Grp78 KO disrupted lung morphogenesis, causing developmental arrest, increased alveolar epithelial type II cell apoptosis, and decreased surfactant protein and type I cell marker expression in perinatal lungs. cGrp78(f/f) pups died immediately after birth, likely owing to respiratory distress. Importantly, Grp78 KO triggered UPR activation with marked induction of the proapoptotic transcription factor CCAAT/enhancer-binding proteins (C/EBP) homologous protein (CHOP). Increased expression of genes involved in oxidative stress and cell death and decreased expression of genes encoding antioxidant enzymes suggest a role for oxidative stress in alveolar epithelial cell (AEC) apoptosis. Increased Smad3 phosphorylation and expression of transforming growth factor-ß/Smad3 targets Cdkn1a (encoding p21) and Gadd45a suggest that interactions among the apoptotic arm of the UPR, oxidative stress, and transforming growth factor-ß/Smad signaling pathways contribute to Grp78 KO-induced AEC apoptosis and developmental arrest. Chemical chaperone Tauroursodeoxycholic acid reduced UPR activation and apoptosis in cGrp78(f/f) lungs cultured ex vivo, confirming a role for ER stress in observed AEC abnormalities. These results demonstrate a key role for GRP78 in AEC survival and gene expression during lung development through modulation of ER stress, and suggest the UPR as a potential therapeutic target in BPD.